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. 2019 Mar 18;9(1):4799.
doi: 10.1038/s41598-019-41264-4.

NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity

Nadine Radomski  1 , Kati Franzke  2 , Svea Matthiesen  1 , Axel Karger  3 , Michael R Knittler  4
Affiliations

NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity

Nadine Radomski et al. Sci Rep. .

Abstract

Natural killer (NK) cells are innate immune cells critically involved in the early immune response against various pathogens including chlamydia. Here, we demonstrate that chlamydia-infected NK cells prevent the intracellular establishment and growth of the bacteria. Upon infection, they display functional maturation characterized by enhanced IFN-γ secretion, CD146 induction, PKCϴ activation, and granule secretion. Eventually, chlamydia are released in a non-infectious, highly immunogenic form driving a potent Th1 immune response. Further, anti-chlamydial antibodies generated during immunization neutralize the infection of epithelial cells. The release of chlamydia from NK cells requires PKCϴ function and active degranulation, while granule-associated granzyme B drives the loss of chlamydial infectivity. Cellular infection and bacterial release can be undergone repeatedly and do not affect NK cell function. Strikingly, NK cells passing through such an infection cycle significantly improve their cytotoxicity. Thus, NK cells not only protect themselves against productive chlamydial infections but also actively trigger potent anti-bacterial responses.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Chlamydial infection of KY-2 cells. (a) KY-2 cells were infected for 24 h in the presence of inhibitors blocking cellular uptake (100 µM DMA, 10 mM MβCD, 200 µM MDC). Lysates were analysed by Western blot probed for chlHSP60 and β-actin. (b,c) Western blots (left) of infected KY-2 and MN-R cells showing chlHSP60 in lysates (intracell.) and culture supernatants (supern.). β-actin served as a loading control. chlHSP60 signals were determined by densitometric analysis (right). chlHSP60 signal intensities in cells (black column part) and supernatants (grey column part) are shown. The signal of total chlHSP60 at 24 hpi was set to 1. (d) Depicts an infected KY-2 cell (24 hpi) stained for chlamydial LPS (green) overlaid on the phase-contrast image. DNA is visualized with DAPI (blue). The size of bacterial structures are indicated by white lines. (e) KY-2 cells were infected or not with chlamydia (0–72 hpi, MOI 40) and stained for chlamydia (green) and DNA (DAPI, blue). A total of 200 cells was counted from 10 random fields of view (63x objective) to determine the number of chlamydial structures in infected NK cells (0–72 hpi). The results are shown in the top panel of (e). Infected KY-2 cells (MOI 40 and 24 hpi) were also co-stained for chlamydia (green), DNA (DAPI, blue), Giantin (Golgi, red) or γ-tubulin (MTOC, red). 55–75% of the KY-2 cells were infected. (a,b,c) Depict cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Figs S4, S5 and S6, respectively.
Figure 2
Figure 2
Chlamydial load of infected KY-2 cells. (a) RT-PCR of chlamydial factors. PCR amplification products were separated on a 1% agarose gel (amplicons of mRNA of gyrA, ftsW, sctN, groEL-1 of chlamydia-infected KY-2 cells (MOI 40)). GAPDH served as control. (b) Flow cytometric analysis of infected KY-2 cells (0–72 hpi). To detect/quantify chlamydia-positive NK cells (green), the negative cell population (black) was identified and gated via corresponding non-infected controls and then subtracted from the total cell population. Flow cytometric analysis of necrotic/apoptotic KY-2 cells during infection was performed by using annexin V-FITC kit from Miltenyi Biotec and propidium iodide (see inserts in Fig. 1b). The original agarose gel image (a) is shown in the Supplementary Fig. S7.
Figure 3
Figure 3
Functional activation of KY-2 cells during chlamydial infection. (a) Flow cytometric analysis of IFN-γ and perforin expression of infected KY-2 cells (MOI 40, 48 hpi). The plot shows the staining intensity of IFN-γ- and perforin-positive KY-2 cells compared with the value for non-infected cells, which was set to 1. Statistical analysis was performed as described in methods (**p < 0.01 and ***p < 0.001 vs. control (non-infected), n = 3). (b) ELISA of IFN-γ secretion of infected KY-2 cells. The plot displays the relative amount of IFN-γ secretion as means ± SD. The maximum value at 72 hpi was set to 3. (c) RT-PCR of CD146 transcript levels in uninfected and infected KY-2 cells (0–72 hpi). Amplicons were separated on a 1% agarose gel. GAPDH served as a control. The plot in (d) shows the relative granzyme B secretion from infected KY-2 cells (MOI 40, 0–72 hpi) measured by ELISA. The values obtained for non-infected cells were set to 1 (*p < 0.05 and ***p < 0.001 vs. control (non-infected), n = 3). (e) Western blot of PKCΘ phospho-activation during KY-2 cell infection (left panel). KY-2 cells were infected or not with chlamydia (MOI 40) for 0–72 h and analysed by Western blots probed for P-PKCϴ, PKCϴ, and chlHSP60. β-actin served as a loading control. After densitometric analysis, the P-PKCϴ/PKCϴ ratio was plotted for the different time points of infection (right panel). (f) Immunofluorescence showing the co-localization between PKCϴ (red) and chlamydia (green) in infected NK cells (MOI 40, 48 hpi). (g) Western blot of chlHSP60 in infected KY-2 cells (MOI 40) and culture supernatants in the presence of sotrastaurin (250 nM). β-actin served as a loading control. (e and g) Depict cropped blots obtained by each protein evaluation. Full-length blots and the original agarose gel image (c) are shown in the Supplementary Figs S8, S9, and S10, respectively.
Figure 4
Figure 4
Chlamydial structures and their intracellular colocalization with secretory granules in infected KY-2 cells. (a) TEM of non-infected (0 hpi, I and IV) and infected (48 and 72 hpi, MOI 40) KY-2 cells (II, III, V and VI). Arrowheads and asterisks indicate chlamydial/granular remnants inside vacuolar structures and on the cell surface (72 hpi). (b) Immunofluorescence of perforin (red, upper panels) or COPI (red, lower panels) and chlamydia (green) in infected (MOI 40, 48 hpi) and non-infected cells. DNA (blue) was stained with DAPI. Cross-reactivity of anti-perforin and anti-α-COP with chlamydia was checked with isolated/purified bacteria (Supplementary Fig. S11). (c) Western blot of chlHSP60 expression in infected KY-2 cells (MOI 40) in the presence of PP2 (0.1 μM) or U73122 (10 μM). Cell lysates were analysed by Western blots probed for chlHSP60 and β-actin. (d) After densitometric analysis, chlHSP60 signals obtained for infected cells (48 hpi) were set to 1 (left: intracellular; right: supernatant). (c) Depicts cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Fig. S12.
Figure 5
Figure 5
Chlamydial infection of primary NK cells. (a) MACS-isolated primary NK cells examined via flow cytometry to determine the proportion of NK1.1-positive cells isolated from the spleen of C57BL/6 mice (dot plot overlay of IgG isotype control (grey) and NK1.1-specific surface staining (green)). (b) Western blot of infected primary NK cells (MOI 40) probed for chlHSP60 in cell lysates (intracell.) and culture supernatants (supern.) (left). After 3 h of infection, extracellular chlamydia were removed by washing with PBS (0 hpi). Cells were further cultivated in fresh medium and lysed at various time points. Corresponding culture supernatants were centrifuged and obtained pellets were also collected. β-actin served as a loading control. chlHSP60 signal intensities (intracell. and supern.) were determined by densitometric analysis (right). The graph shows chlHSP60 in cells (black column part) and supernatants (grey column part). Total chlHSP60 at 24 hpi was set to 1. (c) ELISA of IFN-γ release by infected primary NK cells (MOI 40) (left panel). The plot displays the relative amount of IFN-γ secretion as means ± SD. The maximum value at 72 hpi was set to 3. (d) The plot shows the relative granzyme B secretion in infected primary NK cells (MOI 40, 0–72 hpi) measured by ELISA. The values for non-infected controls were set to 1. Statistical analysis was performed as described in methods (*p < 0.05 and ***p < 0.001 vs. control (non-infected), n = 3). (e) Immunofluorescence demonstrating the co-localization between perforin (red) and chlamydia (green) in infected primary NK cells (MOI 40, 24–72 hpi). (b) Depicts cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Fig. S13.
Figure 6
Figure 6
Pre-infection/recovery/reinfection and its impact on KY-2 cells and chlamydial infectivity. (a) For Western blot, KY-2 cells were pre-infected (MOI 40) for 72 h, washed, recovered for 72 h and then reinfected. At different time points, cells were lysed and pellet fractions of corresponding culture supernatants were collected. β-actin served as a loading control. Primary infection of KY-2 cells is shown in the upper part of (a). (b) Flow cytometry of target cell killing by pre-infected/recovered and/or non-infected KY-2 cells. Adherent KY-2 cells were co-cultured for 4 h with YAC-1 or RMA-S cells with an effector/target ratio (E:T) of 10:1 and 20:1, respectively. The suspension target cells were carefully separated from KY-2 cells by aspiration and stained with PI. For controls, fixed KY-2 cells were mixed with target cells immediately before staining. The graph shows the fold increase of permeabilized target cells after co-cultivation with KY-2 cells compared to the control cell mix (mean values from three measurements ± SD, *p < 0.05 and **p < 0.01 vs. control (non-infected), n = 3). (c) Flow cytometry of the infectivity of culture supernatants (supern.) from infected epithelial and KY-2 cells. KY-2 cells (non-infected and recovered after primary pre-infection) and MN-R cells were infected (MOI 30) or not for 48 h. Culture supernatants were used for incubation with BGM reporter cells. The graph shows the relative amount of chlamydia-positive cells (48 hpi). (d) Flow cytometry of epithelial cell infection after treatment of EBs with granzyme B. EBs were incubated for 4 h at 37 °C with proteolytically activated granzyme B or left untreated. EBs were washed and used for the infection of MN-R cells (48 hpi, MOI 30). The result is depicted as a histogram plot (right panel) (***p < 0.001 vs. control (non-infected), n = 3). (a) depicts cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Fig. S14.
Figure 7
Figure 7
Characterization of the antibody response triggered by KY-2 cell-released chlamydia. (a) C57BL/6 mice were immunized with non-infectious chlamydia from KY-2 cells (controls were treated with PBS). Vaccinated and control sera were analysed in Western blots (a) and immunofluorescence studies (b). Enriched EBs/RBs were used for Western blots (a) The immunostaining (a,b) was performed with control and/or vaccinated serum. Anti-mouse pan-IgG-HRP (a) or -FITC (b) were used as secondary antibodies. Western blots were also probed for chlHSP60. (b) BGM reporter cells were infected or not (MOI 20, 48 hpi) and stained with anti-chlamydial antibody (red), control or vaccinated serum (green). (c) Expression of cytokines/chemokines after immunization with KY-2 cell-released chlamydia (upper panel). Sera from vaccinated and control mice were analysed via cytokine array (R&D Systems). The plot shows the group of cytokines/chemokines with altered levels (mean ± SD, n = 3 independent experiments). For the DC maturation assay (lower panel), primary mouse BMDCs were cultured for 48 h in medium containing 30% serum from vaccinated or control mice and analyzed by flow cytometry. (d) Enriched EBs and RBs were analysed in Western blots, which were first incubated with vaccinated serum and then probed with secondary anti-mouse IgG-HRP antibodies specific for different IgGs. chlHSP60 staining served as a control. (e) Comparability of IgG subclass recognition was checked with purified IgGs. (f) Flow cytometry of BGM reporter cells infected with EBs (48 hpi, MOI 20), which were pretreated (4 h) or not with vaccinated and control serum (non-diluted or diluted). The plot (bottom, left panel) displays the amount of chlamydia-positive cells as means ± SD (n.s.: not significant; **p < 0.01 and ***p < 0.001) vs. control (non-infected), n = 3). (a,d,e) Depict cropped blots obtained by each protein evaluation. Full-length blots are shown in the Supplementary Figs S15, S16 and S17, respectively.
Figure 8
Figure 8
Postulated working model for the anti-chlamydial defence of NK cells and the immune response triggered by the released inactivated non-infectious chlamydia. The depicted model shows the transient chlamydial infection of NK cells triggering activation, cytokine secretion, bacterial granule fusion and chlamydial release (N, nucleus; GA, Golgi apparatus; ER, endoplasmic reticulum; PM, plasma membrane).
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